Recombinant Mouse Forkhead box protein J3 (Foxj3)

What is the basic structural and functional characterization of mouse FOXJ3?

Mouse FOXJ3 is a member of the forkhead box (Fox) family of transcription factors, characterized by a conserved DNA-binding domain called the forkhead domain. The protein contains approximately 622 amino acids and functions primarily as a transcriptional regulator. FOXJ3 contains a forkhead DNA binding region and phosphorylation sites, notably at Ser223 and T216, which play critical roles in its regulatory function .

Western blot analysis shows that FOXJ3 is detected as a band of approximately 80 kDa in nuclear extracts . The protein structure analysis indicates that FOXJ3 binds to DNA through its forkhead domain, which adopts a winged-helix conformation similar to other FOX proteins .

How is FOXJ3 expression regulated in different mouse tissues?

FOXJ3 demonstrates tissue-specific expression patterns, with particularly high expression observed in:

• Spermatogonia and meiotic spermatocytes within mouse testes

• Skeletal muscle tissue

• Neural tissue during development

Expression analysis using conditional knockout models has shown that FOXJ3 expression is temporally regulated during development, with critical expression periods during early postnatal development, especially in reproductive and muscle tissues .

What are the main transcriptional targets of FOXJ3?

FOXJ3 regulates multiple genes involved in various biological processes:

Transcriptional assays in C2C12 myoblasts demonstrated that FOXJ3 activates the Mef2c gene in a dose-dependent manner through binding to a conserved forkhead binding site .

What phenotypes are observed in FOXJ3 knockout mouse models?

Several distinct phenotypes have been observed in FOXJ3 knockout models:

• Muscle-specific effects: FOXJ3 mutant mice have significantly fewer Type I slow-twitch myofibers and impaired skeletal muscle contractile function compared to wild-type controls. Following severe injury, they demonstrate impaired muscle regeneration .

• Reproductive phenotypes:

  • In Foxj3^flox/flox, Mvh-Cre mice (deletion in spermatogonia): Complete male sterility due to Sertoli-cell-only syndrome, with loss of spermatogonia as early as postnatal day 4

  • In Foxj3^flox/flox, Stra8-Cre mice (deletion in spermatocytes): Complete male sterility due to meiotic arrest

• Cellular phenotypes:

  • FOXJ3-deficient spermatogonia show accumulation of DNA double-stranded breaks

  • FOXJ3-deficient myogenic progenitor cells have perturbed cell cycle kinetics

How can I generate and validate recombinant mouse FOXJ3 for experimental use?

Recombinant mouse FOXJ3 can be generated using several approaches:

• E. coli expression system:

  • Clone the mouse FOXJ3 cDNA (full-length or specific domains) into a bacterial expression vector with appropriate tags (His-tag is commonly used)

  • Express in E. coli Rosetta (DE3) strain at optimized conditions

  • Purify using affinity chromatography and validate using Western blot with anti-FOXJ3 antibodies

• Validation methods:

  • Western blot analysis using specific antibodies against FOXJ3 or epitope tags

  • DNA binding assays to confirm functionality (electrophoretic mobility shift assay or chromatin immunoprecipitation)

  • Transcriptional activation assays using reporter constructs containing FOXJ3 binding sites

For optimal activity, consider using the RBR-forkhead domains (rather than forkhead domain alone), as studies have shown that the linker region (RBR) is important for proper folding and DNA binding specificity .

What are the most effective methods for studying FOXJ3 DNA binding specificity?

Several complementary approaches can be used to study FOXJ3 DNA binding specificity:

• Chromatin Immunoprecipitation (ChIP):

  • ChIP-seq analysis has revealed that FOXJ3 binds to regions containing the consensus motif similar to other FOX proteins but with some unique preferences

  • FOXJ3 shows preference for specific nucleotides in regions flanking the core GTAAACA motif

• In vitro DNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) with recombinant FOXJ3 protein and labeled DNA probes

  • DNA footprinting to identify protected regions

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Specialized functional analysis:

  • SEC-MALS (Size Exclusion Chromatography-Multi Angle Light Scattering) to determine protein oligomerization state, which affects DNA binding

  • Domain deletion/mutation analysis to identify critical regions for DNA binding specificity

Research has shown that FOXJ3 RBR-forkhead construct demonstrates different DNA binding preferences compared to the forkhead domain alone, highlighting the importance of the linker region for proper DNA recognition .

How does FOXJ3 contribute to muscle development and regeneration?

FOXJ3 plays critical roles in muscle biology through several mechanisms:

• Myofiber type determination:

  • FOXJ3 mutant mice have significantly fewer Type I slow-twitch myofibers

  • This suggests FOXJ3 is essential for establishing or maintaining the slow-twitch muscle fiber phenotype

• Transcriptional regulation:

  • FOXJ3 directly activates Mef2c, a key transcription factor in muscle development

  • Binds to an evolutionary conserved forkhead binding site (FBS) in the skeletal muscle 5' upstream enhancer of Mef2c

• Muscle regeneration:

  • Following severe injury, FOXJ3 mutant mice exhibit impaired muscle regeneration

  • FOXJ3 mutant myogenic progenitor cells show altered cell cycle kinetics

  • This indicates FOXJ3's importance in the proper function of satellite cells during regeneration

The transcriptional activation of Mef2c by FOXJ3 appears to be dose-dependent and requires binding to the conserved FBS in the enhancer region .

What is the role of FOXJ3 in spermatogenesis and male fertility?

FOXJ3 has stage-specific roles in male germ cell development, as demonstrated by conditional knockout mouse models:

• Role in spermatogonia survival:

  • FOXJ3 deletion in spermatogonia (Foxj3^flox/flox, Mvh-Cre) causes complete male sterility

  • Spermatogonia are lost as early as postnatal day 4

  • The mechanism involves increased DNA double-stranded breaks, suggesting a role in genome stability

• Role in meiotic progression:

  • FOXJ3 deletion in spermatocytes (Foxj3^flox/flox, Stra8-Cre) leads to meiotic arrest

  • Expression of key meiotic proteins (Rad51, Dmc1, Brca1, Brca2, Brit1, Eif4g3, Hop2, Hormad1, and Rnf212) is significantly reduced

  • This indicates FOXJ3 regulates genes essential for meiotic processes

These distinct phenotypes highlight FOXJ3's dual role in both pre-meiotic germ cell survival and the proper execution of meiotic processes.

What is the relationship between FOXJ3 and other FOX family members in regulating transcriptional networks?

FOXJ3 functions within complex transcriptional networks alongside other FOX family members:

• Overlapping genomic binding sites:

  • Genome-wide binding studies reveal extensive overlap between FOXJ3, FOXK2, and FOXO3 binding regions

  • Over 60% of FOXJ3 binding regions are also occupied by FOXK2

  • Approximately 27% of FOXJ3 binding sites overlap with regions co-bound by both FOXK2 and FOXO3

• Differential binding preferences:

  • Despite sharing core consensus motifs, FOX proteins show differences in flanking sequence preferences

  • FOXJ3 and FOXO3 preferentially bind to a variant of the consensus GTAAACA motif that incorporates two A residues preceding the TAAACA sequence

  • These subtle differences contribute to gene-specific regulation

• Functional consequences:

  • Regions bound by multiple FOX proteins (FOXK2/FOXO3/FOXJ3) show higher FOX protein occupancy

  • These regions are enriched for the histone acetylation mark H3K18ac, suggesting active regulatory status

  • Genes associated with these multiply-bound regions are enriched for important biological processes including apoptotic signaling

This indicates that FOX proteins may function through dynamic, partial occupancy of the same sites rather than mutually exclusive binding of individual factors.

What is the role of FOXJ3 in cancer development and progression?

Recent research has revealed associations between FOXJ3 expression and cancer:

Further comprehensive analysis across various cancer types has identified FOXJ3 as a potential prognostic biomarker in specific cancer types.

How do post-translational modifications affect FOXJ3 function in pathological conditions?

Post-translational modifications significantly impact FOXJ3 function in both normal and pathological conditions:

• Phosphorylation patterns:

  • Phosphorylation analysis reveals that the S223 locus of FOXJ3 shows higher phosphorylation levels in breast cancer but decreased levels in pancreatic adenocarcinoma (PAAD) and glioblastoma multiforme (GBM)

  • The T216 locus shows reduced phosphorylation in clear cell renal cell carcinoma (ccRCC) but increased phosphorylation in lung adenocarcinoma (LUAD) and hepatocellular carcinoma

• Functional consequences:

  • Altered phosphorylation may affect FOXJ3's DNA binding capacity, protein-protein interactions, or subcellular localization

  • These changes likely contribute to tissue-specific transcriptional programs in different cancer types

  • The opposing phosphorylation patterns in different cancers suggest context-dependent regulation mechanisms

These findings suggest that FOXJ3 protein phosphorylation at S223 and T216 plays vital roles in cancer development and progression, with different modifications potentially driving distinct pathological outcomes.

How does FOXJ3 compare to FOXP3 in terms of structure and immunoregulatory function?

While both are members of the FOX family, FOXJ3 and FOXP3 exhibit distinct structural features and functional roles:

• Structural differences:

  • FOXP3 forms a unique head-to-head dimer using a linker (Runx1-binding region, RBR) preceding the forkhead domain

  • This head-to-head dimerization confers distinct DNA-binding specificity and creates a docking site for the cofactor Runx1

  • In contrast, FOXJ3 functions through different protein-protein interactions

• Immunoregulatory functions:

  • FOXP3 is the master regulator for regulatory T cell (Treg) development and function

  • FOXP3 controls immune homeostasis, with mutations causing autoimmune diseases

  • Cell-permeable FOXP3 can suppress T helper cell differentiation and alleviate experimental autoimmune conditions

  • In contrast, FOXJ3 has not been directly implicated in immune regulation but rather in tissue development and cellular differentiation

• Therapeutic applications:

  • Protein transduction domain (PTD)-conjugated FOXP3 has been developed as a therapeutic tool for autoimmune conditions

  • PTD-FOXP3 can block Th1 and Th17 differentiation and attenuate inflammatory conditions

  • Similar approaches have not yet been reported for FOXJ3, reflecting their different biological roles

The distinct functions of these FOX family members highlight the specialization that has evolved within this transcription factor family despite their structural similarities.

What are the methodological challenges in generating functional recombinant FOXJ3 protein for structural studies?

Researchers face several technical challenges when producing recombinant FOXJ3 for structural studies:

• Protein solubility and stability issues:

  • Full-length FOXJ3 tends to form aggregates or show poor solubility

  • Domain-specific approaches may be more successful, focusing on the forkhead domain with adjacent regulatory regions

  • The RBR-forkhead construct shows better stability and native-like function compared to the forkhead domain alone

• Proper folding considerations:

  • The RBR linker region is crucial for proper folding of the forkhead domain

  • Truncation of RBR can induce domain-swap dimerization, which may impair function

  • Expression systems that facilitate proper folding (such as eukaryotic expression systems) may be preferred over bacterial systems for full-length protein

• Purification strategies:

  • Fusion tags (NusA, 60 kDa) have been used to improve accuracy of molecular weight estimation by SEC-MALS

  • Multi-step purification approaches combining affinity chromatography, ion exchange, and size exclusion methods are recommended

  • Careful buffer optimization to maintain protein stability during concentration and storage is essential

These considerations are important for generating functionally relevant FOXJ3 protein for both structural studies and functional assays.

How do genomic binding profiles of FOXJ3 change during cellular differentiation or stress conditions?

The dynamic nature of FOXJ3 binding during cellular state changes remains an active area of research:

• Context-dependent binding patterns:

  • Genome-wide binding studies reveal that FOXJ3 binding patterns can change dramatically during cellular differentiation

  • The relationship between FOXJ3 binding and the acquisition of histone marks (such as H3K18ac) suggests a link to changing chromatin states

• Cooperative binding with other factors:

  • FOXJ3 binding is influenced by the presence of other transcription factors

  • During stress conditions, changes in the expression or activity of partner proteins may redirect FOXJ3 to different genomic loci

  • Regions bound by multiple FOX proteins (FOXK2/FOXO3/FOXJ3) show higher regulatory activity

• Methodological approaches to study dynamic binding:

  • Time-course ChIP-seq experiments during differentiation or following stress induction

  • Integration with transcriptome data to correlate binding changes with gene expression

  • Comparison of binding profiles in different cell types or tissues to identify context-specific binding sites

These dynamics likely contribute to the tissue-specific functions of FOXJ3 in development and disease.

How can CRISPR-Cas9 technology be optimized for studying FOXJ3 function in different tissue contexts?

CRISPR-Cas9 technology offers powerful approaches for studying FOXJ3 function:

• Design considerations for tissue-specific studies:

  • Use tissue-specific promoters to drive Cas9 expression

  • Combine with inducible systems (e.g., Tet-On/Off) for temporal control

  • Single or multiple guide RNAs targeting different FOXJ3 functional domains can reveal domain-specific functions

• Delivery methods for different tissues:

  • For muscle studies: AAV-mediated delivery or electroporation into skeletal muscle

  • For reproductive tissue: lentiviral injection into seminiferous tubules

  • For developmental studies: embryonic delivery or generation of conditional alleles

• Functional readouts and validation approaches:

  • Combine with reporter systems to monitor transcriptional activity of FOXJ3 targets

  • Validate editing efficiency using targeted sequencing

  • Confirm functional consequences through analysis of downstream target genes (e.g., Mef2c in muscle, meiotic genes in testes)

• Advanced CRISPR applications:

  • CRISPRa/CRISPRi for modulating FOXJ3 expression without genomic editing

  • Base editors or prime editors for introducing specific mutations in FOXJ3 binding sites

  • CRISPR screening to identify FOXJ3 target genes or interaction partners

These approaches can help overcome the limitations of traditional knockout models by providing greater temporal and spatial control of FOXJ3 function.

What emerging technologies could advance our understanding of FOXJ3 protein-protein interactions?

Several cutting-edge technologies hold promise for elucidating FOXJ3's interactome:

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to FOXJ3 in living cells

  • APEX2-based proximity labeling for temporal mapping of interactions during cellular processes

  • These methods can identify weak or transient interactions that may be missed by traditional co-immunoprecipitation

• Advanced structural biology techniques:

  • Cryo-electron microscopy of FOXJ3-containing complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein interaction surfaces

  • Integrative structural biology combining NMR, X-ray crystallography, and computational modeling

• Single-molecule imaging approaches:

  • Live-cell imaging of fluorescently tagged FOXJ3 to track dynamic interactions

  • Single-molecule FRET to study conformational changes upon partner binding

  • Super-resolution microscopy to visualize FOXJ3 complexes at endogenous loci

These technologies could reveal how FOXJ3 integrates into larger transcriptional complexes and how these interactions are regulated in different cellular contexts.

How might FOXJ3 function be targeted therapeutically in muscle disorders or reproductive pathologies?

Therapeutic approaches targeting FOXJ3 function could be developed for specific pathologies:

• Potential approaches for muscle disorders:

  • AAV-mediated delivery of FOXJ3 to enhance muscle regeneration after injury

  • Small molecule screening to identify compounds that enhance FOXJ3-dependent activation of Mef2c

  • RNA-based therapeutics (antisense oligonucleotides or siRNA) to modulate FOXJ3 levels in specific fiber types

• Strategies for reproductive medicine:

  • Recombinant FOXJ3 protein delivery systems to support spermatogonial stem cell survival in vitro

  • Identification of downstream targets that could bypass FOXJ3 deficiency in male infertility

  • Diagnostic applications to identify FOXJ3 mutations in unexplained male infertility cases

• Delivery challenges and solutions:

  • Tissue-specific targeting strategies using cell-penetrating peptides or nanoparticles

  • Development of small molecule modulators of FOXJ3 activity through structure-based drug design

  • Gene therapy approaches using tissue-specific promoters

These potential therapeutic directions require further basic research to fully understand the tissue-specific functions and regulatory mechanisms of FOXJ3.